Thesis (Ph.D. (Biochemistry, Microbiology & Biotechnology)) - Rhodes University, 2007.

Thesis (Ph.D.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-10-11T22:26:01Z No. of bitstreams: 1 ValeskaSantanaDeSenaPereira_TESE.pdf: 4424545 bytes, checksum: 27d11137997bb962be78ddc99763e2fe (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-10-17T22:34:42Z (GMT) No. of bitstreams: 1 ValeskaSantanaDeSenaPereira_TESE.pdf: 4424545 bytes, checksum: 27d11137997bb962be78ddc99763e2fe (MD5) / Made available in DSpace on 2016-10-17T22:34:42Z (GMT). No. of bitstreams: 1 ValeskaSantanaDeSenaPereira_TESE.pdf: 4424545 bytes, checksum: 27d11137997bb962be78ddc99763e2fe (MD5) Previous issue date: 2016-05-18 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A resist?ncia aos antimal?ricos dispon?veis no mercado leva ? necessidade do desenvolvimento de novos compostos com novos alvos farmacol?gicos. Os derivados de naftoquinonas s?o descritos como compostos l?deres promissores para o desenvolvimento de f?rmacos antimal?ricos. Em vista disso, n?s avaliamos a atividade antiplasmodial in vitro de tr?s derivados de hidroxinaftoquinonas contra o est?gio intraeritroc?tico assexuado de Plasmodium falciparum, assim como par?metros toxicol?gicos in vitro e in vivo e investigamos um prov?vel mecanismo de a??o relacionado ? via dos isopren?ides atrav?s de marca??es metab?licas de precursores da via com tr?tio radioativo, complementado com estudos de docking com um template da octaprenil pirofosfato sintase. Os derivados de hidroxinaftoquinonas analisados tiveram boa atividade antiplasmodial, com IC50 menor que 20 ?M para a cepa 3D7 e menor que 50 ?M para a cepa Dd2. A janela terap?utica ? segura, com ?ndice de seletividade variando entre 36,7 e 143,0. Os compostos n?o causaram hem?lise nas doses testadas (10 e 50 vezes maiores que as respectivas IC50), e n?o desencadearam sinais de toxicidade no teste de toxicidade aguda in vivo apesar de o composto 4a ter promovido esteatose hep?tica e hemorragia no tecido renal. Considerando um prov?vel mecanismo de a??o, os derivados de hidroxinaftoquinonas parecem inibir a s?ntese dos precursores isopr?nicos, principalmente a menaquinona e o tocoferol e os estudos de docking revelaram nove poss?veis intera??es com alta energia em quatro s?tios de liga??o diferentes com um template da octaprenil pirofosfato sintase. Em nossos resultados, o composto 4c foi o mais promissor, visto que possuiu o menor IC50 no teste antiplasmodial in vitro, menor citotoxicidade in vitro e toxicidade aguda in vivo, al?m de ter inibido os tr?s produtos da via dos isopren?ides testados, podendo ser considerado um candidato padr?o para o processo de ?hit-to-lead. / The resistance to antimalarial drugs available on the market leads to the need for the development of new compounds with novel pharmacological targets. The naphthoquinone derivatives are described as promising compounds leading to the development of antimalarial drugs. That said, we evaluated the antiplasmodial in vitro activity of three derivatives of hydroxy-naphthoquinones against asexual intraeritrocitic stage of Plasmodium falciparum, as well as toxicological in vitro and in vivo parameters and investigate a possible mechanism of action related to the isoprenoid pathway through metabolic markers via the precursors of radioactive tritium, complete with docking studies with a template of octaprenil pyrophosphate synthase. Hydroxy-naphthoquinones derivatives analyzed had good antiplasmodial activity with IC50 less than 20 ?M for 3D7 strain and less than 50 ?M for Dd2 strain. The therapeutic window is safe with selectivity index ranging between 36.7 and 143.0. The compounds did not cause hemolysis at the doses tested (10 and 50 times greater than their IC50), and not triggered signs of toxicity in acute toxicity test in vivo even though the compound 4a have promoted hepatic steatosis and haemorrhage in kidney tissue. Whereas a likely mechanism of action, the hydroxy-naphthoquinones derivatives appear to inhibit the synthesis of isoprenic precursors, especially menaquinone and tocopherol and docking studies revealed nine possible interactions with high energy in four different binding sites with a template of octaprenil pyrophosphate synthase. In our results, the compound 4c was the most promising, since it possessed the lowest IC50 in antiplasmodial test in vitro, lower cytotoxicity in vitro and in vivo acute toxicity, and has inhibited the three via the tested isoprenoid products, might be considered a standard candidate for the process "hit-to-lead.

Orientador: Vanderlan da Silva Bolzani / Coorientador : Marilia Valli / Banca: Nivaldo Boralle / Banca: Amanda Danuello Pivatto / Resumo: Na presente pesquisa foram sintetizados vários derivados piridínicos objetivando a descoberta de constituintes com atividade antiparasitária potencial. Estes compostos foram planejados a partir do alcaloide (-)-espectalina isolado de Senna spectabilis (Fabaceae), empregando alguns processos usuais da química medicinal como a simplificação molecular, isosterismo clássico e funcional. Deste procedimento experimental, foram sintetizados 18 substâncias, sendo todas obtidas por meio de reações simples, a partir de compostos comerciais. Das substâncias sintéticas produzidas, 14 foram submetidas à avaliação para atividade em Trypanosoma cruzi, visando potenciais antichagásicos, e em Leishmania infantum para identificar potenciais leishmanicidas. Os compostos testados mostraram-se inativos na inibição destes protozoários. Contudo, dois análogos (48 e 52) apresentaram atividade inibitória das cepas de Plasmodium falciparum, com CI50 = 5,0 µM e índice de seletividade bastante interessante (IS >50). / Abstract: In the present research, several pyridine derivatives were synthesized aiming at the discovery of new constituents with potential antiparasitic activity. All compounds were planned from the (-)-spectaline alkaloid isolated from Senna spectabilis (Fabaceae), employing some usual medicinal chemistry procedures, such as molecular simplification, classical and functional isosterism. From this experimental procedure, 18 compounds were synthesized, and all of these were obtained by means of simple reactions, from commercial compounds. Of the synthetic substances produced, 14 were evaluated for activity in Trypanosoma cruzi, aimed at the search for potential antichagasics, and against Leishmania infantum to identify potential leishmanicidal derivatives. All compounds tested were inactive to the inhibition of these protozoa. However, two analogues (48 and 52) showed inhibitory activity against Plasmodium falciparum strains, with IC50 = 5.0 μM and an interesting selectivity index (IS> 50). / Mestre

Magister Scientiae - MSc / Malaria remains the most important parasitic disease worldwide. It is estimated that over 500 million infections and more that 2.7 million deaths arising from malaria occur each year. Most (90%) of the infections occur in Africa with the most affected groups being children of less than five years of age and women. this dire situation is exacerbated by the emrggence of drug resistant strains of Plasmodium falciparum. The work reported in this thesis focuses on improving the purification of PfHPRT by investigating the characteristics of anion exchange DE-52 chromatography (the first stage of purification), developing an HPLC gel filtration method for examining the quaternary structure of the protein and possible end stage purification, and initialcrystalization trials. a homology model of the open, unligaded PfHPRT is constructed using the atoomic structures of human, T.ccruz and STryphimurium HPRT as templates. / South Africa

Malaria is an infectious disease caused by parasites of the Plasmodium genus with mortality rates of more than a million annually, hence a major global public health concern. Plasmodium falciparum (P. falciparum) accounts for over 90% of malaria incidence. Increased resistance to antimalarial drugs by the Plasmodium parasite, coupled with the lack of an effective malaria vaccine necessitates the urgent need for new research avenues to develop novel and more potent antimalarial drugs. This study focused on falcipains, a group of P. falciparum cysteine proteases that belong to the clan CA and papain family C1, that have emerged as potential drug targets due to their involvement in a range of crucial functions in the P. falciparum life cycle. Recently, falcipain-2 has been validated as a drug target but little is known of its Plasmodium orthologs. Currently, there are several falcipain inhibitors that have been identified, most of which are peptide based but none has proceeded to drug development due to associated poor pharmacological profiles and susceptibility to degradation by host cysteine proteases. Non-peptides inhibitors have been shown to be more stable in vivo but limited information exists. In vivo studies on falcipain-2 and falcipain-3 inhibitors have also been complicated by varying outcomes, thus a good understanding of the structural variations of falcipain Plasmodium orthologs at the active site could go a long way to ease in vivo results interpretation and effective inhibitor design. In this study, we use bioinformatics approaches to perform comparative sequence and structural analysis and molecular docking to characterize protein-inhibitor interactions of falcipain homologs at the active site. Known FP-2 and FP-3 small molecule nonpeptide inhibitors were used to identify residue variations and their effect on inhibitor binding. This was done with the aim of screening a collection of selected non-peptide compounds of South African natural origin to identify possible new inhibitor leads. Natural compounds with high binding affinities across all Plasmodium orthologs were identified. These compounds were then used to search the ZINC database for similar compounds which could have better binding affinities across all selected falcipain homologs. Compounds with high binding affinities across all Plasmodium orthologs were found.

A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.

On a montré que les PTM jouaient un rôle significatif de P. falciparum dans l'année de contrôle de la régulation transcriptionnelle, de l'expression monoaléique et de la différenciation sexuelle. Dix SET contenant des HKMTs contenant du domaine-ont été prédits; Six d'entre eux être essentiels pour le développement de stages de sang asexués. Le projet de thèse est centré sur la caractérisation biologique de PfSET7 et PfSET6. Nous avons observé l'échange de localisation cellulaire dynamique Pendant le cycle de vie: PfSET7 se trouvent dans de multiples foyers cytoplasmiques dans les stades érythrocytaires asexués et le stage de foie, et plus frappante, dans la membrane du parasite enrichie en gamétocytes. PfSET6 EXPOSÉ une localisation nucléaire dans les anneaux et un modèle ponctué dans le cytoplasme des trophozoites matures et schizontes, et est enrichi dans les structures de foyers dans le cytoplasme des gamétocytes. Pris ensemble, notre étude suggère que la méthylation non histone est beaucoup plus significative chez P. falciparum que précédemment attendu. La méthylation à médiation par PfSET7 Peut être une extension du code histone à - d'autres protéines cytosoliques; Partiellement PfSET6 s'associe à des voies de répression transcriptionnelle dans le noyau et des régulateurs post-transcriptionnels dans le cytoplasme. Une étude plus approfondie vise à identifier des cibles de domaine SET contenant des protéines dans le gène inducible knockout mutant parasite lignes. Le fait que PfSET7 et PfSET6 sont exprimés à différents stades du cycle de vie, les fait comme de nouvelles cibles pour le développement de médicaments contre cette maladie grave et de bloquer la transmission. / In P. falciparum, PTMs have been shown to play an important role in the control of transcriptional regulation, monoallelic expression, and sexual differentiation. Ten SET domain-containing HKMTs have been predicted; six of them appear to be essential for asexual blood stage development. My lab has expressed and purified two enzymatically active recombinant methyltransferase PfSET7 and PfSET6. In vitro enzyme kinetics assays shows they can methylate histones. The dissertation project is centered around the biological characterization of PfSET7 and PfSET6. We observed the dynamic changes of cellular localization during life cycle: PfSET7 are found in multiple cytoplasmic foci in asexual erythrocytic stages and liver stage, and more strikingly, enriched in parasite membrane in gametocytes. PfSET6 exhibited a nuclear localization in rings and a punctuated pattern in the cytoplasm of mature trophozoites and schizonts, and is enriched within foci-like structures in the cytoplasm of gametocytes. Taken together, our study suggests that non-histone methylation is much more important in P. falciparum than previously anticipated. PfSET7-mediated methylation may be an extension of the histone code to other cytosol proteins; PfSET6 partially associates with transcriptional repression pathways in the nucleus and post-transcriptional regulators in the cytoplasm. Further study aims to identify targets of SET domain containing proteins within the inducible gene knock out mutant parasite lines. The fact that PfSET7 and PfSET6 are expressed in different life cycle stages, makes them as novel targets for drug development that could against severe disease and to block pathogen transmission.

Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-10T19:15:34Z No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-10T19:15:44Z (GMT) No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-10T19:15:54Z (GMT) No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) / Made available in DSpace on 2015-04-10T19:15:54Z (GMT). No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Belo Horizonte, MG, Brasil / O diagnóstico adequado de malária permanece como um dos pilares dos programas de controle da doença no mundo, já que um diagnóstico eficiente permite a identificação precoce de casos e definição do esquema terapêutico, contribuindo para interrupção do ciclo biológico do parasito. A microscopia óptica (MO), atual diagnóstico de referência para malária, tem apresentado limitações, principalmente em casos de co-infecções e baixas parasitemias. Assim sendo, busca-se por técnicas mais sensíveis e específicas para auxiliar a MO, sendo os métodos baseados na reação em cadeia da polimerase (PCR) considerados mais adequados para identificação de indivíduos com infecção submicroscópica. Entretanto, os vários protocolos de PCR apresentados até o momento tem se baseado no gene da subunidade menor do RNA ribossomal 18S dos plasmódios (18S rRNA), que se encontra em poucas cópias no genoma destes parasitos. Recentemente, foram descritas sequências não ribossomais para identificação de Plasmodium vivax (Pvr47) e Plasmodium falciparum (Pfr364), sendo estas sequências promissoras para o diagnóstico molecular de malária. Baseando-se nestes achados, este trabalho propôs o desenvolvimento de um protocolo de Real-Time PCR para validar os alvos Pvr47 e Pfr364 para o diagnóstico de malária vivax e falciparum, respectivamente, com ênfase em infecções mistas e baixas parasitemias. Após padronização com sucesso da técnica de Real-Time PCR (RT-LAMAL), a mesma foi comparada a três outros protocolos moleculares, sendo duas técnicas baseadas no gene 18S rRNA – Nested-PCR (Snounou et al., 1993) e Real-Time PCR (Mangold et al., 2005) – e uma PCR convencional baseada nos alvos Pvr47/Pfr364 (Demas et al., 2011). Para avaliar os protocolos quanto aos seus limites de detecção de infecções únicas e mistas por P. vivax e P. falciparum, foram realizadas titulações de misturas artificiais com diferentes concentrações dos parasitos. Os resultados obtidos nesta etapa revelaram que a PCR-Demas e RT-LAMAL apresentaram os menores limites de detecção para P. vivax e P. falciparum, tanto em infecções únicas quanto mistas, e que o protocolo de RT-Mangold foi incapaz de detectar coinfecções. Posteriormente, os protocolos foram avaliados para o diagnóstico de malária em amostras de campo, incluindo área não endêmica (n=117) e área endêmica para malária (n=163). Os resultados revelaram que os protocolos de RTMangold, PCR-Demas e RT-LAMAL foram mais eficientes na detecção de parasitemias submicroscópicas, porém, o protocolo de RT-Mangold novamente mostrou ser incapaz de detectar infecções mistas. Em conjunto, os dados obtidos demonstraram que os alvos Pvr47/Pfr364 foram mais adequados para o diagnóstico molecular de malária vivax e falciparum do que o gene 18S rRNA. Contudo, o protocolo aqui desenvolvido (RT-LAMAL) se mostra em vantagem à PCR-Demas, com maior rapidez na obtenção de resultados, dispensando a revelação em gel de agarose e uso de brometo de etídeo, além de diminuir a possibilidade de contaminação de reagentes e amostras. Portanto, conclui-se que a RT-LAMAL possui grande potencial para o diagnóstico molecular de certeza de pacientes com infecções mistas e baixas parasitemias. / Accurate diagnosis of malaria remains as one of the pillars for prevention and control of the disease. An efficient diagnostic test may allow early case detection and appropriate treatment, therefore contributing to the interruption of malaria transmission cycle. Because the optical microscopy (OM) – the gold standard of malaria diagnosis – presents significant limitations, mainly in cases of co-infections and low parasitemias, it seems to be essential to develop a more sensitive and specific diagnostic tool. In this context, methods based on the polymerase chain reaction (PCR) seem to be more suitable for detecting individuals with submicroscopic malaria infections. Unfortunately, the majority of PCR-based methods still rely on the 18S rRNA gene targets, present in few copies in the parasite genome. Recently, new target DNA sequences were described for the identification of Plasmodium vivax (Pvr47) and Plasmodium falciparum (Pfr364). Due to the importance of these findings, the goal of the present study was to validate the Pvr47 and Pfr364 targets for the diagnosis of vivax and falciparum malaria, focusing on the development of a real-time PCR for detection of mixed infection and sub-microscopic parasitemia. After successful standardization of the Real-Time PCR (RT-LAMAL), this-PCR protocol was compared with three well-established PCR protocols, two of them relied on the gene 18S rRNA – Nested-PCR (Snounou et al., 1993) and Real-Time PCR (Mangold et al., 2005) -- and a third protocol based on the Pvr47/Pfr364 as target for a conventional PCR assay (Demas et al., 2011). In order to evaluate these different PCR-protocols in terms of their limit of detection, titrations of artificial mixtures of DNA plasmodial were performed. The results revealed that the PCRDemas and RT-LAMAL presented the lowest limit of detection for either single or mixed P. vivax and P. falciparum infections. In addition, the RT-Mangold protocol was unable to detect co-infections. To further evaluate the performance of these four PCR protocols for diagnosis of malaria in the field, we analyzed samples from malaria-endemic areas (n=163) as well as from non-endemic area (n = 117). While the results confirmed that PCR-Demas and RT-LAMAL protocols as being more appropriate for the diagnosis of submicroscopic infection, the data demonstrated again that the RT-Mangold protocol was unable to detect mixed infections. Together, the data confirmed Pvr47/Pfr364 targets as more suitable for molecular diagnosis of P. vivax and P. falciparum. Of interest, the Real-time PCR developed here (RTLAMAL) offers several advantages over the traditional PCR-Demas, including faster processing time and decreased risk of contamination. In conclusion, that RT-LAMAL has great potential for molecular diagnosis of patients with mixed infections and low levels of parasitemia.

La malaria est la 1ère parasitose mondiale du fait de son taux de morbidité et de mortalité. Elle est responsable de 198 millions de cas dont 584 000 décès en 2013 (OMS). La forme la plus sévère est due à l’apicomplexe Plasmodium falciparum. Etant donné l’absence d’un vaccin efficace et l’augmentation des résistances aux traitements, il est crucial d’approfondir nos connaissances sur la biologie de P. falciparum afin de trouver de nouvelles cibles thérapeutiques. Le cycle de vie complexe avec deux hôtes nécessite une régulation précise et dynamique de l’expression des gènes et des modifications post-traductionnelles. Dans ce contexte, il a été montré que les kinases et les phosphatases, impliquées dans les processus de phosphorylation et de déphosphorylation respectivement, jouent un rôle crucial pour la survie du parasite. Chez les eucaryotes, les phosphatases sont impliquées dans la croissance cellulaire, la différentiation et la division. Parmi elles, PP1, une des principales sérine/thréonine phosphatases, est composée d’une sous-unité catalytique (PP1c) et d’une sous-unité régulatrice. Ces régulateurs sont essentiels et confère à PP1 une localisation, une spécificité et une régulation de son activité. La majorité des régulateurs interagissent avec PP1c via différents motifs tel que le motif RVxF. Chez P. falciparum, PP1 (PfPP1c) est exprimée et semble être essentielle au niveau du stade érythrocytaire, en particulier dans la libération des mérozoïtes infectieux. Pour mieux comprendre la fonction de PfPP1c, nous étudions les régulateurs de PP1 chez le parasite. Nos études précédentes nous ont permis de caractériser 3 régulateurs au niveau moléculaire et fonctionnel. Dans ce contexte, nous avons montré que PfLRR1 et PfI2 inhibent l’activité de PP1 alors que PfI3 l’active. Des études de génétique inverse suggèrent que ces régulateurs sont aussi essentiels que la PP1c elle-même. Récemment, nous avons identifié dans le génome de P. falciparum le facteur d’initiation de la traduction de type 2 sous-unité ß (eIF2ß) qui pourrait être un partenaire/régulateur potentiel de PfPP1. Dans la 1ère partie de cette étude, l’objectif principal a été d’étudier la présence de motifs additionnels de fixation à PfPP1c dans PfI2 et leur impact sur sa fonction. En utilisant la RMN, un troisième motif d’interaction FxxR/KxR/K a été identifié. Ce motif a été montré comme agissant de concert avec le motif canonique RVxF. En effet, la mutation des deux motifs abolie complètement l’interaction avec PfPP1. De plus, en utilisant le modèle d’ovocytes de Xénope, nous avons montré que ces motifs sont nécessaires à PfI2 pour réguler l’activité de PP1. Finalement, l’utilisation d’un peptide dérivé du motif d’interaction FxxR/KxR/K de PfI2 a montré une accumulation dans les érythrocytes infectés et un effet anti-plasmodial a été observé. Dans la 2ème partie de cette étude, nous avons étudié eIF2β, un autre régulateur potentiel de PfPP1. Par des expériences de GST pull-down, nous avons montré l’interaction entre PfeIF2β/PfPP1 et deux motifs d’interaction ont été identifiés : RVxF et FxxR/kxR/K. De plus, en utilisant le modèle d’ovocytes de Xénope, nous avons démontré que PfeIF2ß est impliqué dans la transition G2/M, suggérant un rôle inhibiteur sur l’activité de PP1. La mutation d’un des deux motifs n’empêche pas la formation du complexe alors que la mutation des deux abolie l’interaction avec PP1. Afin de déterminer la fonction de PfeIF2ß in vivo chez Plasmodium, des expériences de génétique inverse ont été réalisées. Nous avons montré l’accessibilité au locus du PfeIF2ß par Knock-in et des expériences d’interruption du gène elf2ß chez Plasmodium falciparum et berghei (espèce spécifique aux rongeurs) sont actuellement en cours afin de déterminer l’essentialité de cette protéine dans le développement du parasite. / Malaria is still the most severe infectious disease in the world because of its high rate of morbidity and mortality. Malaria is responsible for 198 million cases among which 584 000 deaths in 2013 (WHO). The most deadly parasite is the Apicomplexa Plasmodium falciparum. Given the lack of efficient vaccine with long-lasting protection and the increase of resistance against current treatments it is crucial to further deepen our understanding the biology of Plasmodium falciparum to find new means of control. The complex life cycle within two hosts necessitates a highly accurate and dynamic regulation of gene expression and of post translational modifications. In this context, it has been shown that kinases and phosphatases, involved in phosphorylation/dephosphorylation processes respectively, play a key role in parasite survival. In eukaryotes, phosphatases have been shown to be involved in cell growth, differentiation and division. Among them, Protein phosphatase type 1 (PP1) has been reported as one of the major serine/threonine phosphatase proteins involved in diverse cellular functions. PP1 is composed of a single catalytic subunit (PP1c) with a capacity to interact with a high number of regulatory subunits. These regulators are essential as they are key players in different roles of PP1c, including its trafficking, activity and specificity. Most of regulators interact with PP1c via several binding motifs including the RVXF motif. In Plasmodium falciparum, PP1c (PfPP1c) is expressed and seems to be essential for blood stage parasite, in particular merozoïte liberation. To better understand the function of PfPP1c, we investigated the regulators of protein phosphatase type I in this parasite. Our earlier studies have characterized three regulators at the molecular and functional levels. In this context, we have shown that PfLRR1 and PfI2 inhibit PP1 activity while PfI3 activates it. Reverse genetic studies suggested that these regulators are as essential as the PP1c itself. Recently, we found in P. falciparum genome the eukaryotic translation initiation factor 2 subunit ß (eIF2ß) which could be a potential partner/regulator of PfPP1. In the first part of this study, the main objective was to further explore in PfI2 the presence of additional motifs of binding to PfPP1c and their impacts on its function. Using NMR spectroscopy, a third motif was identified: FxxR/KxR/K. This motif has been found to act together with the canonical motif RVxF. Indeed, mutations in both motifs abolished completely the interaction with PfPP1. In addition, using Xenopus oocytes model, we showed that both motifs were necessary for PfI2 to regulate the activity of PP1. Finally the use of a peptide spanning the FxxR/KxR/K motif of PfI2 regulator showed an accumulation in infected erythrocytes and an antiplasmodial effect was observed.In the second part, we investigated eIF2ß as a potential regulator of PfPP1. By GST pull-down assays, we have shown the interaction between PfeIF2ß/PfPP1 and two binding motifs were identified : RVxF and FxxR/KxR/K motifs. Moreover, using Xenopus oocytes model, we demonstrated that PfeIF2ß is involved in G2/M transition, suggesting an inhibitor function of PP1 activity. Mutation of one of two motifs did not prevent the interaction while mutation of both abolished this binding. To gain more insights on the function of PfeIF2ß in Plasmodium, reverse genetic experiments were carried out. We have shown the accessibility of PfeIF2ß locus by Knock-in and we are performing Knock-out experiments on Plasmodium falciparum and berghei (specific species of the rodents) to determine the essentiality of this protein for parasite development.